Bottom Line:
A fully segregating missense mutation (p.A387T) in exon 9 was found in 14 cases and 572 unaffected NEs (pFisher = 3.5×10-27) with a high carrier frequency (25.3%).The mutation interrupts a highly conserved residue in the metalloprotease domain of ADAMTS10, likely affecting its functional capacity.Our study identifies the genetic cause of primary glaucoma in NEs and enables the development of a genetic test for breeding purposes.

Affiliation: Department of Veterinary Biosciences and Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland; The Folkhälsan Institute of Genetics, Helsinki, Finland.

ABSTRACTPrimary glaucoma is one of the most common causes of irreversible blindness both in humans and in dogs. Glaucoma is an optic neuropathy affecting the retinal ganglion cells and optic nerve, and elevated intraocular pressure is commonly associated with the disease. Glaucoma is broadly classified into primary open angle (POAG), primary closed angle (PCAG) and primary congenital glaucoma (PCG). Human glaucomas are genetically heterogeneous and multiple loci have been identified. Glaucoma affects several dog breeds but only three loci and one gene have been implicated so far. We have investigated the genetics of primary glaucoma in the Norwegian Elkhound (NE). We established a small pedigree around the affected NEs collected from Finland, US and UK and performed a genome-wide association study with 9 cases and 8 controls to map the glaucoma gene to 750 kb region on canine chromosome 20 (praw = 4.93×10-6, pgenome = 0.025). The associated region contains a previously identified glaucoma gene, ADAMTS10, which was subjected to mutation screening in the coding regions. A fully segregating missense mutation (p.A387T) in exon 9 was found in 14 cases and 572 unaffected NEs (pFisher = 3.5×10-27) with a high carrier frequency (25.3%). The mutation interrupts a highly conserved residue in the metalloprotease domain of ADAMTS10, likely affecting its functional capacity. Our study identifies the genetic cause of primary glaucoma in NEs and enables the development of a genetic test for breeding purposes. This study establishes also a new spontaneous canine model for glaucoma research to study the ADAMTS10 biology in optical neuropathy.

pone-0111941-g001: Pedigree of glaucoma affected Norwegian Elkhounds.The pedigree constructed around affected dogs indicates a likely recessive mode of inheritance as the affected dogs are born to unaffected parents and there are multiple affected littermates in some litter. The squared dogs were included in the GWAS. Individuals marked with yellow background were genotyped as obligatory carriers and were all heterozygous for the mutation.

Mentions:
A genome-wide association study (GWAS) was performed using Illumina’s CanineHD BeadChip array (San Diego, CA, USA) with 9 cases and 8 controls (Fig. 1). The control dogs were at least 8 years of age without any clinical signs of glaucoma. Genotyping was performed at the Geneseek (Lincoln, NE, USA) and the genotyping data was analyzed using PLINK 1.07 analysis software. A total of 173,662 markers were initially included for the analysis. No individual were removed for low genotyping success of 95%. Missingness test of 95% removed 17,484 SNPs. A total of 72,715 SNPs had minor allele frequency of less than 5% and were removed. None of the SNPs deviated from Hardy-Weinberg equilibrium based of HWE test of P< = 0.0001. After frequency and genotyping pruning, 89,277 SNPs remained in the analysis. A case-controls association test was performed using PLINK software to compare the allele frequencies between cases and controls (Fig. 2A). Identity-by-state (IBS) clustering and CMH meta-analysis (PLINK) were used to adjust for population stratification. Genome-wide corrected empirical p-values were determined applying 100,000 permutations to the data. Besides PLINK the data was analyzed with compressed mixed linear model [32] implemented in the GAPIT R package [33] and with R-implemented GenABEL [34] software (data not shown). The GWAS data is publicly available at dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/).

pone-0111941-g001: Pedigree of glaucoma affected Norwegian Elkhounds.The pedigree constructed around affected dogs indicates a likely recessive mode of inheritance as the affected dogs are born to unaffected parents and there are multiple affected littermates in some litter. The squared dogs were included in the GWAS. Individuals marked with yellow background were genotyped as obligatory carriers and were all heterozygous for the mutation.

Mentions:
A genome-wide association study (GWAS) was performed using Illumina’s CanineHD BeadChip array (San Diego, CA, USA) with 9 cases and 8 controls (Fig. 1). The control dogs were at least 8 years of age without any clinical signs of glaucoma. Genotyping was performed at the Geneseek (Lincoln, NE, USA) and the genotyping data was analyzed using PLINK 1.07 analysis software. A total of 173,662 markers were initially included for the analysis. No individual were removed for low genotyping success of 95%. Missingness test of 95% removed 17,484 SNPs. A total of 72,715 SNPs had minor allele frequency of less than 5% and were removed. None of the SNPs deviated from Hardy-Weinberg equilibrium based of HWE test of P< = 0.0001. After frequency and genotyping pruning, 89,277 SNPs remained in the analysis. A case-controls association test was performed using PLINK software to compare the allele frequencies between cases and controls (Fig. 2A). Identity-by-state (IBS) clustering and CMH meta-analysis (PLINK) were used to adjust for population stratification. Genome-wide corrected empirical p-values were determined applying 100,000 permutations to the data. Besides PLINK the data was analyzed with compressed mixed linear model [32] implemented in the GAPIT R package [33] and with R-implemented GenABEL [34] software (data not shown). The GWAS data is publicly available at dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/).

Bottom Line:
A fully segregating missense mutation (p.A387T) in exon 9 was found in 14 cases and 572 unaffected NEs (pFisher = 3.5×10-27) with a high carrier frequency (25.3%).The mutation interrupts a highly conserved residue in the metalloprotease domain of ADAMTS10, likely affecting its functional capacity.Our study identifies the genetic cause of primary glaucoma in NEs and enables the development of a genetic test for breeding purposes.

Affiliation:
Department of Veterinary Biosciences and Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland; The Folkhälsan Institute of Genetics, Helsinki, Finland.

ABSTRACTPrimary glaucoma is one of the most common causes of irreversible blindness both in humans and in dogs. Glaucoma is an optic neuropathy affecting the retinal ganglion cells and optic nerve, and elevated intraocular pressure is commonly associated with the disease. Glaucoma is broadly classified into primary open angle (POAG), primary closed angle (PCAG) and primary congenital glaucoma (PCG). Human glaucomas are genetically heterogeneous and multiple loci have been identified. Glaucoma affects several dog breeds but only three loci and one gene have been implicated so far. We have investigated the genetics of primary glaucoma in the Norwegian Elkhound (NE). We established a small pedigree around the affected NEs collected from Finland, US and UK and performed a genome-wide association study with 9 cases and 8 controls to map the glaucoma gene to 750 kb region on canine chromosome 20 (praw = 4.93×10-6, pgenome = 0.025). The associated region contains a previously identified glaucoma gene, ADAMTS10, which was subjected to mutation screening in the coding regions. A fully segregating missense mutation (p.A387T) in exon 9 was found in 14 cases and 572 unaffected NEs (pFisher = 3.5×10-27) with a high carrier frequency (25.3%). The mutation interrupts a highly conserved residue in the metalloprotease domain of ADAMTS10, likely affecting its functional capacity. Our study identifies the genetic cause of primary glaucoma in NEs and enables the development of a genetic test for breeding purposes. This study establishes also a new spontaneous canine model for glaucoma research to study the ADAMTS10 biology in optical neuropathy.